High-molecular weight conjugate of steroids

ABSTRACT

Enzymes in the body vary among different species, and also vary among individuals of the same species. Thus, it has been demanded to develop a novel steroid-containing pharmaceutical preparation which can release a drug in a manner independent of the enzymes present in the body, and which is expected to have an efficacious therapeutic effect. 
     Disclosed is a high-molecular weight conjugate of a steroid, comprising a structure in which a carboxylic acid group of a polymer having a polyethylene glycol moiety and a succinic acid monoamide moiety having two or more succinic acid monoamide units is ester-bonded to a hydroxy group in the steroid.

TECHNICAL FIELD

The present invention relates to a high-molecular weight conjugate ofsteroids in which a carboxylic acid group of a polymer having apolyethylene glycol moiety and a succinic acid monoamide moiety isester-bonded to a hydroxy group in the steroids, a method for producingthe same, and the use thereof.

BACKGROUND ART

Steroid pharmaceuticals are a therapeutic agent for various disordersincluding many inflammatory disorders such as rheumatism and collagendisease, allergic diseases, serious infections and cancer, and they haveexcellent therapeutic effects on these disorders. However, when they areprescribed for a certain disorder, various actions of steroids aremanifested as side effects on normal sites other than lesions to betreated, and as a result a dosage amount may be limited in some cases.In addition, it is easily expected that the longer it remains on alesion, the more efficacious the steroid would be on the lesion.However, due to the concern regarding the side effect as describedabove, the number of repeated doses may be also limited. In other words,although the steroids are an excellent pharmaceutical compound whichexhibits efficacy in treating various disorders, the use of steroidstends to be avoided at clinical settings due to their side effects.Currently, as an attempt to solve this problem, studies, for example, toaccumulate the steroids selectively on a lesion and to release them atthe lesion over a long period of time are carried out.

One of such studies relates to a drug delivery system, and as an exampleof the system, Limethasone (manufactured by Mitsubishi PharmaCorporation), which comprises palmitic acid ester of dexamethasone in alipid sphere is known in the art (Adv. Drug Delivery Rev., vol. 20, p.195 (1996)).

Patent Document 1 describes a formulation comprising nano particles ofphosphoric acid ester of betamethasone formed with zinc, polylacticacid, and polyethylene glycolpolylactic acid.

Meanwhile, Patent Document 2 discloses a polymer compound comprising adrug bonded to a block copolymer of polyethylene glycol and polyasparticacid, which forms micelles and has water solubility. Patent Document 3describes a polymer derivative of camptothecins in which a side chaincarboxylic acid group of a block copolymer of polyethylene glycol andpolyglutamic acid is bonded to a phenolic hydroxy group ofcamptothecins. It is known that these polymer compounds accumulate intumors due to an EPR effect. However, neither Patent Document 2 norPatent Document 3 discloses a polymer compound to which steroids arebonded.

-   [Patent Document 1] Japanese Patent Application Laid-open    Publication (Kohyo) No. 2006-521367-   [Patent Document 2] Japanese Patent Publication No. 2694923-   [Patent Document 3] WO 2004/39869-   [Non-Patent Document 1] Adv. Drug Delivery Rev., vol. 20, p. 195    (1996)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Limethasone described above releases palmitic acid ester ofdexamethasone from lipid spheres, and then steroids produced byhydrolysis of the ester bond by enzymes in the body exhibit theiractivity. Similarly, the nano particles described in Patent Document 1release phosphoric acid ester of betamethasone from the particles, andthen steroids produced by hydrolysis of the ester bond by enzymes in thebody exhibit their activity. However, it has been known that activitiesof hydrolyzing enzymes in the body vary not only among different speciesbut also among individuals of the same species, and thus there is also aconcern that the effect of the drug release from the conjugate would begreatly different among individuals when the cleavage of the bond todrug depends on the hydrolyzing enzymes.

In the case of the adriamycin conjugate described in Patent Document 2in which a block copolymer is bonded to adriamycin via an amide bond,the efficacy is questionable since the release of the drug by hydrolysisis slow due to the amide bond, a chemically stable bonding form.

Although steroids including prednisolone, methyl prednisolone,dexamethasone, betamethasone, clobetasol, diflorasone, diflucortolone,fluocinolone acetonide, hydrocortisone, difluprednate, beclometasone,triamcinolone and alclometasone are a useful drug for treating disorderssuch as rheumatism, asthma, nephritis. ulcerative colitis, autoimmunediseases, allergy, psoriasis, eczema, stomatits, granuloma and malignantlymphoma, the side effects are very frequently manifested. Thus, therehas been a demand for a novel drug which does not depend on hydrolyzingenzymes present in the body and delivers the steroids only to desiredsite to manifest fewer side effects.

Means for Solving the Problems

The inventors of the present invention noticed the higher permeabilityof blood vessels around cancer lesions or inflammation sites, and, as aconsequence of studies to solve the problems described above. weconsidered that, as result of the circulation in the body of a drugadministered in the vessels, the drug would be more easily accumulatedin cancer lesions or inflammation sites around which the vessels arehighly permeable. Consequently, they achieved an invention of ahigh-molecular weight conjugate of steroids in which the steroids arechemically bonded to a polymer carrier of a block copolymer includingpolyethylene glycol. The high-molecular weight conjugate of steroids canalso be used as a prodrug.

Efficacy of conventional prodrugs may be likely to vary amongindividuals as the hydrolyzing enzymes of a patient are used for therelease of the drug. On the other hand, the present invention, byintroducing a moiety of succinic acid monoamide bonded to steroids intoa polymer, intends to chemically hydrolyze the conjugate underphysiological condition. Specifically, the inventors found a phenomenonthat a compound having a hydroxy group is easily released as thestructure of succinic acid monoamide changes to a cyclized structure(i.e., succinic imide) when the compound having a hydroxy group isester-bonded to a free carboxylic acid of succinic acid monoamide, andattempted to apply the phenomenon to a block copolymer includingpolyethylene glycol. As a result, they have found that, in thehigh-molecular weight derivative of steroids in which a polymer having apolyethylene glycol moiety and a succinic acid monoamide moiety isester-bonded to a hydroxy group of the steroids having a hydroxy group,the ester bond can be cleaved without depending on hydrolyzing enzymesto release the steroids that are the active entities and to exhibit thepharmacological effects and have completed the present invention.

Specifically, the present invention is related to the following (1) to(14).

(1) A high-molecular weight conjugate of steroids, comprising astructure in which a carboxylic acid group of a polymer having apolyethylene glycol moiety and a succinic acid monoamide moiety havingtwo or more succinic acid monoamide units is ester-bonded to a hydroxygroup in the steroids.

(2) The high-molecular weight conjugate of steroids according to (1)above, wherein the polymer having a polyethylene glycol moiety and asuccinic acid monoamide moiety is a block copolymer.

(3) The high-molecular weight conjugate of steroids according to (1) or(2) above, wherein the succinic acid monoamide moiety is polyasparticacid.

(4) The high-molecular weight conjugate of steroids according to any oneof (1) to (3) above represented by formula (I)

wherein R1 represents a hydrogen atom or a (C1-C6) alkyl group; R2represents a linking group; R3 represents a hydrogen atom or a (C1-C6)acyl group; R4 represents a residue of the hydroxy group of the —OR4represents an alcoholic hydroxy group of said steroids; R5 represents agroup selected from the group consisting of a (C1-C30) alkoxy group, a(C7-C30) aralkyloxy group, a (C1-C30) alkylamino group, a di(C1-C30)alkylamino group, an amino acid with a protected carboxy group and—N(R6)CONH(R7) wherein R6 and R7, which may be identical or differentfrom each other, represent a (C3-C6) cyclic alkyl group or a (C1-C5)alkyl group which may be substituted with a tertiary amine group; trepresents an integer from 5 to 11500; and d, e, f, g, h, i or j eachindependently represents an integer from 0 to 200; provided that d+erepresents an integer from 1 to 200, d+e+f+g+h+i+j represents an integerfrom 3 to 200; and respective units of the polyaspartic acid are bondedin any order.

(5) The high-molecular weight conjugate of steroids according to (4)above, wherein R1 represents a (C1-C6) alkyl group, R2 represents a(C2-C6) alkylene group, R3 represents a (C1-C6) acyl group, t representsan integer of 100 to 300, and d, e, f, g, h, i or j each independentlyrepresents an integer from 0 to 100, provided that d+e represents aninteger from 1 to 100 and d+e+f+g+h+i+j represents an integer from 6 to100.

(6) The high-molecular weight conjugate of steroids according to (5)above, wherein R1 represents a (C1-C3) alkyl group, R2 represents a(C2-C4) alkylene group, R3 represents a (C1-C3) acyl group, t representsan integer from 100 to 300, and d, e, f, g, h, i or j each independentlyrepresents an integer from 0 to 90, provided that d+e represents aninteger from 1 to 90 and d+e+f+g+h+i+j represents an integer from 15 to90.

(7) The high-molecular weight conjugate of steroids according to any oneof (1) to (6) above, in which the steroids are prednisolone, methylprednisolone, dexamethasone, betamethasone, clobetasol, diflorasone,diflucortolone, fluocinolone acetonide, hydrocortisone, difluprednate,beclometasone, triamcinolone or alclometasone.

(8) A high-molecular weight conjugate of steroids which is obtained byester-bonding a carboxylic acid group of a polymer having a polyethyleneglycol moiety and a succinic acid monoamide moiety having two or moresuccinic acid monoamide units to a hydroxy group in the steroids havinghydroxy groups by using a dehydrating condensation agent in an organicsolvent.

(9) A method of manufacturing the high-molecular weight conjugate ofsteroids described in any one of (1) to (7) above, the method comprisingester-bonding a carboxylic acid group of a polymer having a polyethyleneglycol moiety and a succinic acid monoamide moiety having two or moresuccinic acid monoamide units to a hydroxy group of the steroids havingthe hydroxy group, by using a dehydrating condensation agent in anorganic solvent.

(10) A pharmaceutical composition comprising as an active ingredient thehigh-molecular weight conjugate of steroids according to any one of (1)to (8) above.

(11) An anti-inflammatory agent comprising as an active ingredient thehigh-molecular weight conjugate of steroids according to any one of (1)to (8) above.

(12) An anti-rheumatism agent comprising as an active ingredient thehigh-molecular weight conjugate of steroids according to any one of (1)to (8) above.

(13) An agent for treating collagen disease comprising as an activeingredient the high-molecular weight conjugate of steroids according toany one of (1) to (8) above.

(14) An anti-allergy agent comprising as an active ingredient thehigh-molecular weight conjugate of steroids according to any one of (1)to (8) above.

Effect of the Invention

The high-molecular weight conjugate of steroids of the present inventioncan deliver the steroids only to a desired site and release the steroidswithout depending on hydrolyzing enzymes in the body. Thus, withoutbeing affected by difference among individuals, the steroids areexpected to achieve efficacious therapeutic effects.

BEST MODE FOR CARRYING OUT THE INVENTION

The high-molecular weight conjugate of steroids of the present inventionis characterized in that a carboxylic acid group of a polymer having apolyethylene glycol moiety and a succinic acid monoamide moiety havingtwo or more succinic acid monoamide units is ester-bonded to a hydroxygroup in the steroids.

According to the present invention, the term “succinic acid monoamidemoiety” refers to the structure of —HNCO—C—C—CO₂H, and examples thereofinclude succinic acid monoamide (—HNCO—CH₂—CH₂—CO₂H) and a structure inwhich one of the two carboxylic acid groups of aspartic acid is amidated(—HNCO—CH(—NH—)—CH₂—CO₂H or —HNCO—CH₂—CH(—NH—)—CO₂H). These succinicacid monoamide moieties may constitute a polymer backbone, for example,as in the case of polyaspartic acid, or may be bound to a functionalgroup of the backbone polymer composed of a polyalcohol such as dextran,a polyamine such as polylysine, or a polycarboxylic acid other thanpolyaspartic acid (for example, polylactic acid, etc.).

Examples of the polymer having a polyethylene glycol moiety and asuccinic acid monoamide moiety having two or more succinic acidmonoamide units include a graft-type polymer in which the polyethyleneglycol moiety and the succinic acid monoamide moiety branch from thepolymer backbone in a comb-like form, and a block-type polymer (blockcopolymer) in which the polymers having a polyethylene glycol moiety anda succinic acid monoamide moiety are tandemly aligned.

When the succinic acid monoamide moiety forms polyaspartic acid, thegraft-type polymer also includes a polymer in which the polyethyleneglycol moiety is partially bonded to the polyaspartic acid backbone. Theblock-type polymer includes a polymer in which the terminal ofpolyaspartic acid is bonded to the terminal of polyethylene glycolmoiety.

The polyethylene glycol moiety in the polymer of the high-molecularweight conjugate of steroids of the present invention includespolyethylene glycol in which both terminals or a single terminal ismodified. When both terminals are modified, the modifying groups can beidentical or different from each other. Examples of the modifying groupinclude a (C1-C6) alkyl group optionally having a substituent group.Examples of the alkyl group of the (C1-C6) alkyl group optionally havinga substituent group include an alkyl group set forth as R1 in Formula(I) below. Preferred is a (C1-C4) alkyl group including, for example, amethyl group, an ethyl group, an n-propyl group and an n-butyl group.Examples of a substituent group included in the (C1-C6) alkyl groupoptionally having a substituent group include, for example, an aminogroup, a methylamino group, a dimethylamino group, an ethylamino groupand a diethylamino group.

The molecular weight of the polyethylene glycol moiety is about 300 to500,000, preferably about 500 to 100,000, more preferably about 1,000 to50,000.

The molecular weight of the polymer having a polyethylene glycol moietyand a succinic acid monoamide moiety is about 500 to 600,000, preferablyabout 600 to 110,000, more preferably about 800 to 80,000.

According to the present invention, the term “molecular weight” refersto the weight average molecular weight determined by the GPC method.

In the high-molecular weight conjugate of steroids of the presentinvention, the amount of the steroids bonded to the polymer having apolyethylene glycol moiety and a succinic acid monoamide moiety havingtwo or more succinic acid monoamide units is 1 to 100%, preferably 1 to90%, more preferably 2 to 60%, based on the total number of carboxylicacid groups.

According to the present invention, the steroids are not particularlylimited, provided that they are the steroids having an alcoholic hydroxygroup. Examples the steroids include prednisolone represented by thefollowing formula (II), methyl prednisolone represented by the followingformula (III), dexamethasone represented by the following formula (IV),betamethasone represented by the following formula (V), clobetasolrepresented by the following formula (VI), diflorasone represented bythe following formula (VII), diflucortolone represented by the followingformula (VIII), fluocinolone acetonide represented by the followingformula (IX), hydrocortisone represented by the following formula (X), adeacylated product of difluprednate represented by the following formula(XI), beclometasone represented by the following formula (XII),triamcinolone represented by the following formula (XIII) andalclometasone represented by the following formula (XIV). Preferably,examples of the hydroxy group of steroids include, for example, aprimary hydroxy group of prednisolone represented by the followingformula (II) or a secondary hydroxy group of clobetasol represented bythe following formula (VI). However, the position for substitution isnot limited.

The steroids indicated above and derivatives thereof are commerciallyavailable as an anti-allergic agent, an anti-inflammatory agent, anagent for treating autoimmune diseases, an anti-tumor agent, etc.

According to the present invention, the succinic acid monoamide moietyhaving two or more succinic acid monoamide units is preferablypolyaspartic acid.

A preferred high-molecular weight conjugate of steroids of the presentinvention includes a compound represented by formula (I) above, whereinR1 represents a hydrogen atom or a (C1-C6) alkyl group; R2 represents alinking group; R3 represents a hydrogen atom or a (C1-C6) acyl group; R4represents a residue of the hydroxy group of the —OR4 represents analcoholic hydroxy group of said steroids; R5 represents a group selectedfrom the group consisting of a (C1-C30) alkoxy group, a (C7-C30)aralkyloxy group, a (C1-C30) alkylamino group, a di(C1-C30) alkylaminogroup, an amino acid with protected carboxy group and —N(R6)CONH(R7)wherein R6 and R7, which may be identical or different from each other,represent a (C3-C6) cyclic alkyl group or a (C1-C5) alkyl group whichmay be substituted with a tertiary amine group; t represents an integerfrom 5 to 11,500; and d, e, f, g, h, i or j each independentlyrepresents an integer from 0 to 200; provided that d+e represents aninteger from 1 to 200 and that d+e+f+g+h+i+j represents an integer from3 to 200.

Examples of the (C1-C6) alkyl group for R1 in the general formula (I)include a straight-chain or branched (C1-C6) alkyl group, including amethyl group, an ethyl group, an n-propyl group, an i-propyl group, ann-butyl group and a t-butyl group. Preferred is a straight-chain orbranched (C1-C4) alkyl group, and particularly preferred is astraight-chain or branched (C1-C3) alkyl group including a methyl group,an ethyl group, an n-propyl group, and an i-propyl group, and moreparticularly preferred is a methyl group.

Examples of the linking group represented by R2 in the general formula(I) include, but are not particularly limited to, a (C2-C6) alkylenegroup. Preferred is a (C2-C4) alkylene group including, for example, anethylene group, a trimethylene group and a tetramethylene group, andparticularly preferred is a trimethylene group.

Examples of the (C1-C6) acyl group for R3 in the general formula (I)include, but not particularly limited to, a formyl group, an acetylgroup, a propionyl group and a pivaloyl group. Preferred is a (C1-C3)acyl group, and more preferred is an acetyl group.

With regard to the residue of the hydroxy group of the steroids for R4alcoholic hydroxy group of the steroids for —OR4 in the general formula(I), examples of the steroids include the steroids enumerated above.

R5 in the general formula (I) represents a group selected from the groupconsisting of a (C1-C30) alkoxy group, a (C7-C30) aralkyloxy group, a(C1-C30) alkylamino group, a di(C1-C30) alkylamino group, an amino acidwith protected carboxy group, and —N(R6)CONH(R7) wherein R6 and R7,which may be identical or different from each other, represent a (C3-C6)cyclic alkyl group or a (C1-C5) alkyl group which may be substitutedwith a tertiary amino group. R5 in the general formula (I) may beidentical or different from each other in one molecule, and a polymer inthe high-molecular weight conjugate of steroids may include asingle-type R5 or mixed type R5.

A substituent group can be introduced to R5 as necessary. Physicalproperties of the high-molecular weight conjugate of steroids can becontrolled by the introduction of the substituent groups to R5. Forexample, the release rate of the steroids can be freely controlled.

Examples of the (C1-C30) alkoxy group include a straight-chain orbranched (C1-C30) alkoxy group, and preferred is a straight-chain orbranched (C1-C10) alkoxy group, including, for example, a methoxy group,an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxygroup and a t-butoxy group. Examples of the (C7-C30) aralkyloxy groupinclude a straight-chain or branched (C7-C30) aralkyloxy group, andpreferred is a straight-chain or branched (C7-C12) aralkyloxy group,including, for example, a 4-phenylbutoxy group.

Examples of the (C1-C30) alkylamino group or di(C1-C30) alkylamino groupinclude a straight-chain or branched (C1-C30) alkylamino group or astraight-chain or branched di(C1-C30) alkylamino group, and preferred isa straight-chain or branched (C1-C20) alkylamino group or astraight-chain or branched di(C1-C20) alkylamino group, including, forexample, an methylamino group, an ethyl amino group, an n-propylaminogroup, an i-propylamino group, an n-butylamino group, a t-butylaminogroup, a dimethylamino group, a diethylamino group and a dibutylaminogroup.

Examples of the amino acid with protected carboxy group include an aminoacid generally used in peptide synthesis, in which a carboxyl group isprotected, including, for example, a phenylalanine benzyl ester.

Examples of the group —N(R6) CONH(R7) for R5 in the general formula (I),wherein R6 and R7, which may be identical or different from each other,represent a (C3-C6) cyclic alkyl group or a (C1-C5) alkyl groupoptionally substituted with a tertiary amine group include. but notparticularly limited to, for example, acyclohexylaminocarbonylcyclohexylamino group and anisopropylaminocarbonylisopropylamino group.

Polyaspartic acid which is a succinic acid monoamide moiety in thehigh-molecular weight conjugate of steroids represented by the generalformula (I) of the present invention, includes constituent units ofα-amino acid type, β-amino acid type and cyclized type. Theseconstituent units are bound in any order, and may be bound to form ablock-type form or a random-type form.

The total number of aspartic acid residues in the high-molecular weightconjugate of steroids represented by the aforementioned general formula(I) is represented by “d+e+f+g+h+i+j”, and is about 3 to 200, preferablyabout 6 to 100, more preferably about 15 to 90.

The proportion of the number of aspartic acid residues bonded to thesteroids (d+e) based on the total number of aspartic acid residues(d+e+f+g+h+i+j) is 1 to 100%, preferably 3 to 90%, more preferably 4 to60%. In addition, the number of aspartic acid residues (d+e) is about 1to 200, preferably about 1 to 100, more preferably about 1 to 90.

The proportion of α-amino acid type (d+f+h) based on the total number ofaspartic acid residues (d+e+f+g+h+i+j) is 10 to 100%, preferably 20 to100%. The proportion can be appropriately changed, for example, bysuitably selecting a deprotection condition for a protecting group inthe polyaspartic acid which was produced by using the protecting group.

In the aforementioned general formula (I), t is an integer from about 5to 11,500, preferably an integer from about 8 to 2300, more preferably,an integer from about 100 to 300.

The high-molecular weight conjugate of steroids of the present inventionrepresented by the aforementioned general formula (I) may form micelleswith the polyethylene glycol moiety as the outer shell in water.

The high-molecular weight conjugate of steroids of the present inventionis obtained by ester-bonding a carboxylic acid group of a polymer havinga polyethylene glycol moiety and a succinic acid monoamide moiety havingtwo or more succinic acid monoamide units to a hydroxy group of steroidsby using a dehydrating condensation agent in an organic solvent, and thepresent invention also includes this manufacturing method; that is, amanufacturing method of subjecting, for example, a block copolymer of apolyethylene glycol moiety-polyaspartic acid produced by the methoddescribed in Japanese Patent Application Laid-Open No. 6-206815, andsteroids in which the functional groups other than the group to bereacted are protected as necessary, to a reaction using a dehydratingcondensation agent such as dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIPC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt (WSC) and1-ethoxycarbonyl-2-ethoxy-1,2-dihydroxyquinolinone (EEDQ) at atemperature of 0 to 180° C., preferably 5 to 50° C. in an organicsolvent in which both of the block copolymer and the steroids aredissolved, preferably in an aprotic polar solvent such asN,N-dimethylformamide (DMF), 1,3-dimethyl-2-imidazolidinone (DMI) andN-methylpyrrolidone (NMP). Furthermore, a reaction aid such asN,N-dimethylaminopyridine (DMAP) may also be used for the condensationreaction. After condensation reaction, deprotection is carried out asnecessary, and conventional operations for separation and purification,etc., are applied to obtain a high-molecular weight conjugate ofsteroids.

In addition, a high-molecular weight conjugate of steroids in which R5is a —N(R6)CONH(R7) group wherein R6 and R7, which may be identical ordifferent from each other, represent a (C3-C6) cyclic alkyl group or a(C1-C5) alkyl group optionally substituted with a tertiary amine groupmay also be obtained by a reaction using the aforementionedcarbodiimides as an condensation agent.

As a method of introducing as R5 a (C1-C30) alkoxy group, a (C7-C30)aralkyloxy group, a (C1-C30) alkylamino group, a di(C1-C30) alkylaminogroup or an amino acid with protected carboxy group into a compound ofthe general formula (I), there can be mentioned a method in which acarboxylic acid of the polymer is first activated according to theaforementioned method, and then reacted with a corresponding alcohol, acorresponding amine or an amino acid with protected carboxy group in aan amount to be introduced under basic conditions; and a method in whicha corresponding alcohol, a corresponding amine or an amino acid with aprotected carboxy group is first activated, and then reacted with apolymer. After purification of the polymer, it is also possible tore-activate any unreacted carboxylic acid groups of the polymer by thesame reaction, and the re-activated carboxylic acid groups may becondensed with the hydroxyl group of the steroids. Alternatively,different alcohols, amines, etc. are repeatedly reacted to synthesize amixture of polymers having various substituents as R5, with which thehydroxy group of steroids may subsequently condensed. Furthermore, aftercondensation of steroids, a (C1-C30) alkoxy group, a (C7-C30) aralkyloxygroup, a (C1-C30) alkylamino group, a di(C1-C30) alkylamino group or anamino acid with protected carboxy group may be introduced.

However, the method of manufacturing the high-molecular weight conjugateof steroids of the present invention is not intended to be limited tothe aforementioned methods.

The high-molecular weight conjugate of steroids of the present inventionreleases steroids under the condition in the body to exhibit thepharmacological activity of the steroids, and therefore functioning as adrug, it can be used as an anti-inflammatory agent, an anti-rheumatismagent, an agent for treating collagen disease, an anti-allergic agent,an anti-cancer agent, etc. The high-molecular weight conjugate ofsteroids of the present invention can be used in a dosage form which isconventionally used, including, for example, injections, tablets,powders and the like. For formulation process, a pharmaceuticallyacceptable carriers which are conventionally used, for example, bindingagents, lubricating agents, disintegrating agents, solvents, vehicles,solubilizing agents, dispersing agents, stabilizing agents. suspendingagents, preservatives, soothing agents, colorants and flavors can alsobe used. Among various dosage forms, the use as an injection ispreferred, and usually, for example, water, physiological saline, 5%glucose or mannitol solution, water-soluble organic solvent (forexample. glycerol, ethanol, dimethylsulfoxide, N-methylpyrrolidone,polyethylene glycol, Cremophor and the like, or a mixture thereof), or amixture of water and water-soluble organic solvents can be used.

The dosage of the high-molecular weight conjugate of steroids of thepresent invention can varies as a matter of course, depending on sex,age, physiological conditions, pathology and the like of patients. It isparenterally administered, typically at a does of 0.01 to 500 mg/m²,preferably 0.1 to 250 mg/m², as an active ingredient per day.Administration by injection is carried out intravenously,intraarterially, to the affected site (inflammation site), for example.

EXAMPLES

Hereinafter, the present invention will be illustrated more specificallywith reference to Examples, but is not intended to be limited to theExamples. Conditions for analysis by HPLC (high performance liquidchromatography) is as follows:

column: ODS (inertsil ODS-3 4.6×150 mm)

detection: UV 254 nm

elution: A) 0.1% aqueous phosphate solution,

B) acetonitrile/1% aqueous phosphate solution (9/1)

B %=30%.

Example 1

Synthesis of Compound 1 (conjugate of prednisolone and a block copolymerconsisting of methoxypolyethylene glycol moiety having molecular weightof 12,000 and polyaspartic acid moiety having a polymerization number of33: in the general formula (I), R1=Me (methyl group), R2=trimethylenegroup, R3=Ac (acetyl group), R4=prednisolone residue —OR4=the alcoholichydroxy group of prednisolone, R5=isopropylaminocarbonylisopropylaminogroup, d+e+f+g+h+i+j=33, t=273)

An N-acetylated product of a copolymer of polyethylene glycol withmethoxy at one terminal-polyaspartic acid (a block copolymer consistingof a methoxypolyethylene glycol moiety having a molecular weight of12,000 and a polyaspartic acid moiety having a polymerization number of33, and in the general formula (I), R1=Me (methyl group),R2=trimethylene group, R3=Ac (acetyl group), d=e=f=g=j=0; 265 mg)produced according to the production method described in Japanese PatentApplication Laid-open No. 6-206815, and commercially availableprednisolone (manufactured by Tokyo Chemical Industry Co., Ltd.; 100 mg)were dissolved in DMF (3.7 ml), and DMAP (7 mg) and DIPC (0.2 ml) wereadded thereto. The mixture was stirred for 40 hrs at 15° C. To thereaction solution, ethanol (5.6 ml), ethyl acetate (5.6 ml) anddiisopropyl ether (45 ml) were added, and the mixture was stirred for 3hrs at room temperature. Subsequently, the precipitate was collected byfiltration and washed with ethanol/diisopropyl ether (1/4 (v/v); 10 ml).The resulting precipitate was dissolved in acetonitrile/water (1/1(v/v); 20 ml), and then the solution was applied to a column of an ionexchange resin (DOWEX50 (H⁺), manufactured by The Dow Chemical Company;3 ml), and then eluted with acetonitrile/water (1/1 (v/v); 6 ml). Afterwater (20 ml) was added to the eluted fraction thus obtained,acetonitrile was distilled off under reduced pressure, and then theresidue was freeze-dried to obtain Compound 1 (340 mg).

On the basis of the amount of unreacted prednisolone present in thereaction liquid determined by HPLC (high performance liquidchromatography), the content of prednisolone in Compound 1 wasdetermined as 25.4% (w/w), and the proportion of d+e based ond+e+f+g+h+i+j was determined as 44%. In Compound 1, free prednisolonewas not detected.

According to this method, an isopropylaminocarbonyl-isopropylamino groupcan be added as R5, and the abundance ratio of the group was determinedby ¹H-NMR (hydrogen nuclear magnetic resonance spectrum) using Compound1 dissolved in sodium deuteroxide/deuterium oxide/deuteratedacetonitrile. The proportion of isopropylaminocarbonylisopropylaminogroup to polyaspartic acid of Compound 1, that is, the proportion of f+gbased on d+e+f+g+h+i+j was 9%. The remaining aspartic acid residues arein the form of a free carboxylic acid (corresponding to h+i) or a cyclicstructure (corresponding to j).

Example 2

Synthesis of Compound 2 (conjugate of dexamethasone and a blockcopolymer consisting of a methoxypolyethylene glycol moiety having amolecular weight of 12,000 and a polyaspartic acid moiety having apolymerization number of 35: in the general formula (I), R1=Me (methylgroup), R2=trimethylene group, R3=Ac (acetyl group), R4=dexamethasoneresidue —OR4=the alcoholic hydroxy group of dexamethasone,R5=isopropylaminocarbonylisopropylamino group, d+e+f+g+h+i+j=35, t=273)

An N-acetylated product of a copolymer of polyethylene glycol withmethoxy at one terminal-polyaspartic acid produced according to themanufacturing method described in Japanese Patent Application Laid-openNo. 6-206815 (a polymerization number of aspartic acid is 35; 100 mg)and commercially available prednisolone (manufactured by Tokyo ChemicalIndustry Co., Ltd.; 42 mg) were dissolved in DMF (2.0 ml), and DMAP in aDMF solution (0.773 mmol/ml solution, 27.7 μl) and DIPC (67.0 μl) wereadded thereto. The mixture was stirred for 43 hrs at 15° C. DIPC (17.0μl) was further added and stirred for 4 hrs at 30° C. To the reactionsolution, ethyl acetate (4.0 ml) and diisopropyl ether (16 ml) wereadded, and the mixture was stirred for 3 hrs at room temperature.Subsequently, the precipitate was collected by filtration and washedwith ethyl acetate/diisopropyl ether (1/4 (v/v); 10 ml). The resultingprecipitate was dissolved in acetonitrile/water (1/1 (v/v); 12 ml), andthen the solution was applied to a column of an ion exchange resin(DOWEX50 (H⁺), manufactured by The Dow Chemical Company; 10 ml) andeluted with acetonitrile/water (1/1 (v/v); 20 ml). Acetonitrile in theeluted fraction thus obtained was distilled off under reduced pressure,and the residue was freeze-dried to obtain Compound 2 (108 mg).

Compound 2 was collected and treated with alkali to cleave the bondeddexamethasone. By HPLC (high performance liquid chromatography), thecontent of dexamethasone in Compound 2 was determined as 20.7% (w/w) andthe proportion of d+e based on d+e+f+g+h+i+j was determined as 31%. Inthe isolated Compound 2, free dexamethasone was found to be 0.3% (w/w).

Example 3

Synthesis of Compound 3 (conjugate of dexamethasone and a blockcopolymer consisting of a methoxypolyethylene glycol moiety having amolecular weight of 12,000 and a polyaspartic acid moiety having apolymerization number of 35 to which phenylalanine benzyl ester isfurther bonded: in the general formula (I), R1=Me (methyl group),R2=trimethylene group, R3=Ac (acetyl group), R4=dexamethasone residue—OR4=the alcoholic hydroxy group of dexamethasone, R5=phenylalaninebenzyl ester and isopropylaminocarbonylisopropylamino group,d+e+f+g+h+i+j=35, t=273)

An N-acetylated product of methoxypolyethylene glycol with methoxy atone terminal-polyaspartic acid copolymer (polymerization number ofaspartic acid 35; 100 mg) prepared according to the manufacturing methoddescribed in Japanese Patent Application Laid-open No. 6-206815 andcommercially available dexamethasone (manufactured by Tokyo ChemicalIndustry Co., Ltd.; 42 mg) were dissolved in DMF (2.0 ml), and DMAP in aDMF solution (0.773 mmol/ml solution, 27.7 μl) and DIPC (33.5 μl) wereadded thereto. The mixture was stirred for 20 hrs at 15° C. To thereaction solution, a DMF solution of phenylalanine benzyl ester (0.612mmol/ml, 140.0 μl) and DIPC (33.5 μl) were further added and stirred for23 hrs at 30° C. Thereafter, DIPC (17.0 μl) was further added, and themixture was stirred for 4 hrs at 30° C. To the reaction liquid, ethylacetate (4.0 ml) and diisopropyl ether (16 ml) were added. Afterstirring for 3 hrs at room temperature, the precipitate was collected byfiltration and washed with ethyl acetate/diisopropyl ether (1/4 (v/v);10 ml). The resulting precipitate was dissolved in acetonitrile/water(1/1 (v/v); 12 ml), and the solution was applied to a column of an ionexchange resin (DOWEX50 (H⁺), manufactured by The Dow Chemical Company;10 ml), and then eluted with acetonitrile/water (1/1 (v/v); 20 ml).Acetonitrile in the eluted fraction thus obtained was distilled offunder reduced pressure, and the residue was freeze-dried to obtainCompound 3 (112 mg).

Compound 3 was collected and treated with alkali to cleave the bondeddexamethasone. By HPLC (high performance liquid chromatography), thecontent of dexamethasone in Compound 3 was determined as 20.5% (w/w). Inthe isolated Compound 3, free dexamethasone was found to be 0.2% (w/w).

Comparative Example 1

Synthesis of Comparative compound 1 (conjugate of prednisolone and ablock copolymer consisting of methoxypolyethylene glycol moiety havingmolecular weight of 12,000 and polyglutamic acid moiety having apolymerization number of 23)

An N-acetylated product of polyethylene glycol with methoxy at oneterminal-polyglutamic acid copolymer (a block copolymer consisting of amethoxypolyethylene glycol moiety having a molecular weight of 12,000and a polyglutamic acid moiety having a polymerization number of 23; 128mg) prepared according to the manufacturing method described in JapanesePatent Application Laid-Open publication No. 5-955 and commerciallyavailable prednisolone (50 mg) were dissolved in DMF (1.3 ml), and DMAP(2.4 mg) and DIPC (0.06 ml) were added thereto. The mixture was stirredfor 20 hrs at 25° C. To the reaction solution, ethanol (2 ml), ethylacetate (2 ml) and diisopropyl ether (12 ml) were added, and the mixturewas stirred for 30 minutes at room temperature. Subsequently, theprecipitate was collected by filtration and washed withethanol/diisopropyl ether (1/4 (v/v); 2 ml). The resulting precipitatewas dissolved in acetonitrile/water (1/1 (v/v); 7 ml) and applied to acolumn of anion exchange resin (DOWEX50 (H⁺), manufactured by The DowChemical Company; 1 ml), and then eluted with acetonitrile/water (1/1(v/v); 2 ml). After water (10 ml) was added to the eluted fraction thusobtained, acetonitrile was distilled off under reduced pressure, andthen the residue was freeze-dried to obtain Comparative compound 1 (160mg).

On the basis of the amount of unreacted prednisolone present in thereaction solution determined by HPLC (high performance liquidchromatography), the content of prednisolone in Comparative compound 1was determined as 25.8% (w/w). In the isolated Comparative compound 1,no free prednisolone was detected.

Test Example Drug Release in the Absence of Enzymes

Compound 1 or Comparative compound 1, or Compound 2 or Compound 3 wasdissolved in PBS (phosphate buffered physiological saline; pH 7.1) to apolymer concentration of 1 mg/ml, and the solution was incubated at 37°C. Prednisolone or dexamethasone released from Compound 1, Compound 2,Compound 3 or Comparative compound 1 was separated and quantified byHPLC in comparison with a standard curve. The proportion of thequantified value based on the total amount of the drug determined fromthe drug content in the high-molecular weight conjugate is shown inFIGS. 1 and 2.

As shown in FIG. 1, the high-molecular weight conjugate of the presentinvention (Compound 1) released almost 98% of prednisolone within 24 hrseven in the absence of hydrolyzing enzymes. However, Comparativecompound 1 including no succinic acid monoamide moiety hardly releasedprednisolone even after 24 hrs.

As shown in FIG. 2, the high-molecular weight conjugate of the presentinvention (Compounds 2 and 3) was found to be capable of releasingalmost 60 to 90% of dexamethasone within 24 hrs even in the absence ofhydrolyzing enzymes. It was also shown that the linking of phenylalaninebenzyl ester to the high-molecular weight conjugate allowed to controlthe release rate of dexamethasone.

These results demonstrate the excellent drug release performance of thehigh-molecular weight conjugate of steroids of the present inventionunder a neutral condition even in the absence of enzymes, and thus acapability of the high-molecular weight conjugate of steroids of thepresent invention to release steroid under a physiological conditionobserved, for example, in blood or body fluids. It is furtherdemonstrated that, the appropriate linking of a substituent groupincluding, for example, phenylalanine benzyl ester, allows to controlthe release rate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a proportion of the amount of the releasedprednisolone based on the total amount of the prednisolone in theconjugate in PBS solution (pH 7.1; 37° C.) containing Compound 1 of thepresent invention (the high-molecular weight conjugate in whichprednisolone is bonded to a polyaspartic acid moiety) or Comparativecompound 1 (the high-molecular weight conjugate in which prednisolone isbonded to a polyglutamic acid moiety). In FIG. 1, -●- indicates theproportion of the drug released from Compound 1 of the presentinvention, and -∘- indicates the proportion of the drug released fromComparative compound 1.

FIG. 2 illustrates a proportion of the amount of the releaseddexamethasone based on the total amount of the dexamethasone in theconjugate in PBS solution (pH 7.1; 37° C.) containing Compound 2 of thepresent invention (the high-molecular weight conjugate in whichdexamethasone is bonded to a polyaspartic acid moiety) or Compound 3 ofthe present invention (the high-molecular weight conjugate in whichdexamethasone and phenylalanine benzyl ester are bonded to apolyaspartic acid moiety). In FIG. 2, -▴- indicates the proportion ofthe drug released from Compound 2 of the present invention, and -▪-indicates the proportion of the drug released from Compound 3 of thepresent invention.

The invention claimed is:
 1. A high-molecular weight conjugate ofsteroids, comprising a structure in which a carboxylic acid group of ablock copolymer having a polyethylene glycol moiety and polyasparticacid is ester-bonded to an alcoholic hydroxy group in the steroids, andwherein the conjugate is represented by formula (I)

wherein R1 represents a (C1-C6) alkyl group; R2 represents a (C2-C6)alkylene group; R3 represents a (C1-C6) acyl group; R4 —OR4 representsan alcoholic hydroxy group of said steroids; R5 represents a groupselected from the group consisting of a (C1-C30) alkoxy group, a(C7-C30) aralkyloxy group, a (C1-C30) alkylamino group, a di(C1-C30)alkylamino group, an amino acid with a protected carboxy group and—N(R6)CONH(R7) wherein R6 and R7, which may be identical or differentfrom each other, represent a (C3-C6) cyclic alkyl group or a (C1-C5)alkyl group which may be substituted with a tertiary amine group; trepresents an integer from 100 to 300; and d, e, f, g, h, i or j eachindependently represents an integer from 0 to 100; provided that d+erepresents an integer from 1 to 100, d+e+f+g+h+i+j represents an integerfrom 6 to 100; and respective units of the polyaspartic acid are bondedin any order.
 2. The high-molecular weight conjugate of steroidsaccording to claim 1, wherein R1 represents a (C1-C3) alkyl group, R2represents a (C2-C4) alkylene group, R3 represents a (C1-C3) acyl group,t represents an integer from 100 to 300, and d, e, f, g, h, i or j eachindependently represents an integer from 0 to 90, provided that d+erepresents an integer from 1 to 90 and d+e+f+g+h+i+j represents aninteger from 15 to
 90. 3. The high-molecular weight conjugate ofsteroids according to claim 1 or 2, in which the steroids areprednisolone, methyl prednisolone, dexamethasone, betamethasone,clobetasol, diflorasone, diflucortolone, fluocinolone acetonide,hydrocortisone, difluprednate, beclometasone, triamcinolone oralclometasone.
 4. A method of manufacturing the high-molecular weightconjugate of steroids described in claim 1, the method comprisingester-bonding a carboxylic acid group of a block copolymer having apolyethylene glycol moiety and polyaspartic acid to an alcoholic hydroxygroup of the steroids, by using a dehydrating condensation agent in anorganic solvent.
 5. A pharmaceutical composition comprising as an activeingredient the high-molecular weight conjugate of steroids according toclaim
 1. 6. An anti-inflammatory agent comprising as an activeingredient the high-molecular weight conjugate of steroids according toclaim
 1. 7. An anti-rheumatism agent comprising as an active ingredientthe high-molecular weight conjugate of steroids according to claim
 1. 8.An agent for treating collagen disease comprising as an activeingredient the high-molecular weight conjugate of steroids according toclaim
 1. 9. An anti-allergy agent comprising as an active ingredient thehigh-molecular weight conjugate of steroids according to claim
 1. 10.The high-molecular weight conjugate of steroids according to claim 2, inwhich the steroids are prednisolone, methyl prednisolone, dexamethasone,betamethasone, clobetasol, diflorasone, diflucortolone, fluocinoloneacetonide, hydrocortisone, difluprednate, beclometasone, triamcinoloneor alclometasone.